1. Field of the Invention
The present invention relates to a purified, thermostable DNA polymerase purified from the hyperthermophilic eubacteria Thermotoga maritima, and means for isolating and producing the enzyme. Thermostable DNA polymerases are useful in many recombinant DNA techniques, especially nucleic acid amplification by the polymerase chain reaction (PCR).
2. Background Art
Huber et al., 1986, Arch. Microbiol. 144:324-333 describe the isolation of the bacterium Thermotoga maritima. Thermotoga maritima is a eubacterium which is strictly anaerobic, rod-shaped, fermentative, hyperthermophilic and grows between 55.degree. C. and 90.degree. C., with an optimum growth temperature of about 80.degree. C. This eubacterium has been isolated from geothermally heated sea floors in Italy and the Azores.
Thermotoga maritima cells have a sheath-like structure and monotrichous flagellation. Thermotoga maritima is classified in the eubacterial kingdom by visine of its having murein and fatty acid-containing lipids, its diphtheria-toxin-resistant elongation factor 2, its RNA polymerase subunit pattern and its sensitivity to antibiotics.
Extensive research has been conducted on the isolation of DNA polymerases from mesophilic microorganisms such as E. coli. See, for example, Bessman et al., 1957, J. Biol. Chem. 223:171-177, and Buttin and Kornberg, 1966, J. Biol. Chem. 241:5419-5427. Much less investigation has been made on the isolation and purification of DNA polymerases from thermophiles such as Thermotoga maritima. Kaledin et al., 1980, Biokhymiya 45:644-651, discloses a six-step isolation and purification procedure for DNA polymerase from cells of Thermus aquaticus YT-1 strain. These steps involve isolation of crude extract, DEAE-cellulose chromatography, fractionation on hydroxyapatite, fractionation on DEAE-cellulose, and chromatography on single-strand DNA-cellulose. The molecular weight of the purified enzyme is reported by Kaledin et al. as 62,000 daltons per monomeric unit.
A second purification scheme for a polymerase from Thermus aquaticus is described by Chien et at., 1976, J. Bacteriol. 127:1550-1557. In this process, the crude extract is applied to a DEAE-Sephadex column. The dialyzed pooled fractions are then subjected to treatment on a phosphocellulose column. The pooled fractions are dialyzed and bovine serum albumin (BSA) is added to prevent loss of polymerase activity. The resulting mixture is loaded on a DNA-cellulose column. The pooled material from the column is dialyzed. The molecular weight of the purified protein is reported to be about 63,000 daltons to 68,000 daltons.
The use of thermostable enzymes, such as those prepared by Chien et al. and Kaledin et al., to amplify existing nucleic acid sequences in amounts that are large compared to the amount initially present was described U.S. Pat. Nos. 4,683,195 and 4,683,202, which describe the PCR process, both disclosures of which are incorporated herein by reference. Primers, template, nucleoside triphosphates, the appropriate buffer and reaction conditions, and polymerase are used in the PCR process, which involves denaturation of target DNA, hybridization of primers, and synthesis of complementary strands. The extension product of each primer becomes a template for the production of the desired nucleic acid sequence. The two patents disclose that, if the polymerase employed is a thermostable enzyme, then polymerase need not be added after every denaturation step, because heat will not destroy the polymerase activity.
U.S. Pat. No. 4,889,818, European Patent Publication No. 258,017, and PCT Publication No. 89/06691, the disclosures of which are incorporated herein by reference, all describe the isolation and recombinant expression of an .about.94 kDa thermostable DNA polymerase from Thermus aquaticus and the use of that polymerase in PCR. Although T. aquaticus DNA polymerase is especially preferred for use in PCR and other recombinant DNA techniques, there remains a need for other thermostable polymerases.
Accordingly, there is a desire in the art to produce a purified, thermostable DNA polymerase that may be used to improve the PCR process described above and to improve the results obtained when using a thermostable DNA polymerase in other recombinant techniques, such as DNA sequencing, nick-translation, and even reverse transcription. The present invention helps meet that need by providing recombinant expression vectors and purification protocols for Thermotoga maritima DNA polymerase.